Iridoid Glycosides from Lagotis alutacea

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Iridoid Glycosides from Lagotis alutacea

Li-Juan Yangâ, Na Zhangâ, Na Liuâ, and Xiao-Dong Yang^b

aKey Laboratory of Ethnic Medicine Resource Chemistry, State Ethnic Affairs Commission &

Ministry of Education, Yunnan Minzu University, Kunming, 650500, P. R. China

bKey Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming, 650091, P. R. China Reprint requests to Prof. Li-Juan Yang and Prof. Xiao-Dong Yang. Fax: +86-871-65910017.

E-mail:ljyyang@gmail.com(L. J. Yang),xdyang@ynu.edu.cn(X. D. Yang) Z. Naturforsch.2014,69b, 835 – 838 / DOI: 10.5560/ZNB.2014-4093 Received April 25, 2014

One new iridoid glycoside, lagotisoside F (1), was isolated from Lagotis alutacea, along with three known analogs, lagotisoside D (2), 6-O-α-L-(4⁰⁰-O-(E)-cinnamoyl) rhamnopyranosyl-catapol (3) and globularin (4). Their structures were elucidated by extensive spectroscopic analysis and by comparison with data reported in the literature. Compounds1–4were evaluated for cytotoxic activity against HL-60, MCF-7, A549, SW480, and SMMC-7721 cells and exhibited no appreciable activity with IC₅₀values above 40µM.

Key words: Logatis alutacea, Iridoid Glycosides, Lagotisoside F

Introduction

The genus Lagotis (Scrophulariaceae) is repre- sented with 17 species in China, mostly growing in the southwestern part of the country on mountains of 3000 meters above sea level or higher [1]. Sev- eral species such as L. alutacea, L. glauca, L. yun- nanesis, and L. brachystachya have long been used in Tibetan folk medicine for the treatment of fever, high blood pressure, and acute and chronic hepati- tis [2,3]. In the literature, the chemical composi- tions of two species of Lagotis have been studied.

Flavonoids were isolated from L. brachystachya [4], while phenylpropanoid glycosides and iridoid glycosides were found inL. stolonifera[5]. In our efforts to seek structurally interesting compounds from medicinal plants growing on the Yunnan-Tibet Plateau, we investigated the chemical constituents ofL. yunnanesis, and have found a series of iridoid glycosides [6–8].

As part of continued investigations on the genusLago- tis,L. alutaceahas been examined. To the best of our knowledge, no phytochemical study onL. alutaceahas been reported as yet. As a result, one new iridoid glycoside, lagotisoside F (1), was isolated from this plant, together with three known analogs, lagotisoside

D (2) [8], 6-O-α-L-(4⁰⁰-O-(E)-cinnamoyl) rhamnopy- ranosylcatapol (3) [9] and globularin (4) [10] (Fig.1).

Their structures were elucidated by extensive spectroscopic analysis and by comparison with data reported in the literature. Herein, we report on the isolation and structural elucidation of the new iridoid glycoside 1 from this plant.

Results and Discussion

Lagotisoside F (1) was isolated as a colorless amorphous powder,[α]²⁴_D =−168.50 (c=0.345, MeOH).

Its molecular formula was determined as C32H42O17

by HR-FAB-MS (found 698.2425, calcd. 698.2422).

The IR spectrum showed characteristic absorptions of OH (3375 cm⁻¹, br), of an α,β-unsaturated ester (1710 and 1635 cm⁻¹) and an aromatic-ring (1598 and 1508 cm⁻¹). The UV absorptions at 222 (3.59), 303 (3.18) and 312 (3.74) nm also confirmed the presence of these unsaturated functional groups.

The ¹H NMR spectrum (Table1) of 1 indicated the presence of a catalpol unit (H-1, H-3 to H-10, H-1⁰ to H-6⁰) combined with a rhamnose unit (H- 1⁰⁰ to H-6⁰⁰) and a 3,4-disubstituted-(E)-cinnamoyl moiety (H-2⁰⁰⁰, H-5⁰⁰⁰, H-6⁰⁰⁰, H-7⁰⁰⁰, and H-8⁰⁰⁰). The

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836 L.-J. Yanget al.·Iridoid Glycosides fromLagotis alutacea

Fig. 1. The structures of compounds1–5.

two olefinic protons of the cinnamoyl moiety at δ =7.66 (d,J=15.6 Hz, H-7⁰⁰⁰) and δ =6.47 ppm (d, J = 15.6 Hz, H-8⁰⁰⁰) with a coupling constant of 15.6 Hz indicated trans-configuration of the dou- ble bond. An anomeric proton at δ =5.08 (d, J= 2.0 Hz, H-1⁰⁰) and a methyl group at δ =1.33 ppm (d, J=6.0 Hz, H-6⁰⁰) in the ¹H NMR spectrum, as well as ¹³C NMR signals at δ =97.8 (C-1⁰⁰) and 18.2 ppm (C-6⁰⁰) suggested the presence of a rhamnose moiety. Based on the coupling constant of the anomeric proton (J=2.0 Hz), an α-rhamnose was confirmed. In the case of a β-rhamnose, the coupling constant normally is approximately 4.2 Hz [11].

The position of the 3,4-disubstituted-(E)-cinnamoyl moiety was determined by comparison of the ¹H and¹³C NMR spectra with those of unsubstituted 6- O-α-L-rhamnopyranosylcatalpol (5) [12] (Fig.1, Ta- ble1). The C-2⁰⁰ signal of1was shifted downfield by 1.9 ppm; the H-2⁰⁰signal was also shifted downfield by 1.96 ppm, whereas the signals of H-3⁰⁰, H-4⁰⁰ and H- 5⁰⁰were shifted downfield by 0.11, 0.01 and 0.02 ppm, respectively. These features were compatible with the attachment of the acyl group to the C-2 of rhamnose.

This assignment was confirmed by the HMBC spectrum.

In the HMBC spectrum (Fig.2), the correlations of δ_H=5.20 (H-2⁰⁰) toδ_C=168.6 (C-9⁰⁰⁰) confirmed that

Fig. 2. The key HMBC and NOESY correlations of lagotisoside D (1).

the 3,4-disubstituted-(E)-cinnamoyloxy moiety was substituted at the C-2⁰⁰position of rhamnose. The correlations ofδH=4.81 (H-10) toδC=95.27 (C-1) suggested thatβ-D-glucose was substituted at C-1, while δH =5.08 (H-1⁰⁰) to δC =84.3 (C-6) indicated that α-L-rhamnose was substituted at C-6. The correlations betweenδH=3.86 (OCH₃) toδC=150.69 (C- 3⁰⁰⁰) and ofδH=3.86 (OCH₃) toδC=152.87 (C-4⁰⁰⁰) suggested an (E)-configuration of the 3,4-dimethoxy-

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L.-J. Yanget al.·Iridoid Glycosides fromLagotis alutacea 837 (E)-cinnamoyl moiety. NOESY experiments were also

conducted, and the key correlations are shown in Fig.2. The correlations between H-1 and H-6, H-1 and H-7, H-6 and H-7, as well as H-5 and H-9 suggested that the relative configurations of C-1, C-6, C- 7, C-5, and C-9 in compound1are identical to that of catalpol [10].

Therefore, the structure of compound 1was elucidated as 6-O-α-L-(2⁰⁰-O-E-3⁰⁰⁰,4⁰⁰⁰-dimethoxycinma- moyl)rhamnopyranosylcatalpol, named lagotisoside F.

Compounds 1–4 were evaluated in vitro against a panel of human tumor cell lines, including leukemia (HL-60), breast carcinoma (MCF-7), lung carcinoma (A549), colon carcinoma (SW480), and myeloid liver carcinoma (SMMC-7721). The compounds lacked ac- tivities against all tumor cell lines investigated at the concentration of 40µM.

No. 1 5

δH δC δH δC

1 5.09 (d, 1H,J=8.9) 95.3 (d) 5.07 (d, 1H,J=8.9) 95.2 (d) 3 6.39 (dd, 1H,J=6.0, 1.8) 142.4 (d) 6.35 (dd, 1H,J=6.0, 1.8) 142.2 (d) 4 5.07 (dd, 1H,J=6.0, 5.5) 103.6 (d) 5.05 (dd, 1H,J=6.0, 5.0) 103.6 (d)

5 2.44 (m, 1H) 37.3 (d) 2.40 (m, 1H) 37.4 (d)

6 4.02 (dd, 1H,J=8.0, 2.0) 84.3 (d) 3.99 (dd, 1H,J=8.0, 2.0) 83.7 (d) 7 3.64 (d, 1H,J=2.0) 59.6 (d) 3.62 (d, 1H,J=2.0) 59.4 (d)

8 66.7 (s) 67.0 (s)

9 2.58 (dd, 1H,J=9.2, 8.0) 43.3 (d) 2.54 (dd, 1H,J=9.2, 8.0) 43.4 (d) 10 3.83 (d, 1H,J=13.2) 61.5 (t) 3.81 (d, 1H,J=13.0) 61.5 (t)

4.17 (d, 1H,J=13.2) 4.13 (d, 1H,J=13.0)

1⁰ 4.81 (d, 1H,J=8.0) 99.8 (d) 4.77 (d, 1H,J=8.0) 99.8 (d) 2⁰ 3.30 (dd, 1H,J=9.0, 8.0) 74.9 (d) 3.25 (dd, 1H,J=9.0, 8.0) 74.9 (d) 3⁰ 3.45 (dd, 1H,J=9.0, 8.0) 77.7 (d) 3.40 (dd, 1H,J=9.0, 8.0) 77.8 (d) 4⁰ 3.28 (dd, 1H,J=10.0, 8.0) 71.7 (d) 3.24 (dd, 1H,J=10.0, 8.0) 71.8 (d)

5⁰ 3.32 (m, 1H) 78.6 (d) 3.30 (m, 1H) 78.7 (d)

6⁰ 3.61 (dd, 1H,J=12.0, 6.0) 63.0 (t) 3.61 (dd, 1H,J=12.0, 6.0) 63.0 (t) 3.97 (dd, 1H,J=12.0, 2.0) 3.91 (dd, 1H,J=12.0, 2.0)

1⁰⁰ 5.08 (d,J=2.0) 97.8 (d) 4.92 (d,J=2.0) 100.4 (d)

2⁰⁰ 5.20 (dd, 1H,J=3.0, 2.0) 74.2 (d) 3.84 (dd, 1H,J=3.0, 2.0) 72.3 (d) 3⁰⁰ 3.78 (dd, 1H,J=9.0, 3.0) 70.6 (d) 3.67 (dd, 1H,J=9.0, 3.0) 72.4 (d) 4⁰⁰ 3.37 (t, 1H,J=10.0) 71.5 (d) 3.38 (t, 1H,J=10.0) 73.9 (d)

5⁰⁰ 3.63 (m, 1H) 70.3 (d) 3.63 (m, 1H) 71.9 (d)

6⁰⁰ 1.33 (d, 3H,J=6.3) 18.2 (d) 1.30 (d, 3H,J=6.3) 17.9 (q)

1⁰⁰⁰ 128.7 (s)

2⁰⁰⁰ 7.20 (m, 1H) 111.4 (d)

3⁰⁰⁰ 150.7 (s)

4⁰⁰⁰ 152.9 (s)

5⁰⁰⁰ 6.95 (d, 1H,J=8.4) 112.6 (d) 6⁰⁰⁰ 7.15 (d, 1H,J=8.4) 124.4 (d) 7⁰⁰⁰ 7.71 (d, 1H,J=15.6) 147.2 (d) 8⁰⁰⁰ 6.47 (d, 1H,J=15.6) 116.2 (d)

9⁰⁰⁰ 168.6 (s)

MeO-3⁰⁰⁰ 3.85 (s, 3H) 56.6 (q)

MeO-4⁰⁰⁰ 3.86 (s, 3H) 56.6 (q)

Table 1.¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectral data for 1 and 5 (CD3OD, TMS,δ in ppm,J in Hz).

Conclusion

In summary, one new iridoid glycoside, lagotisoside F (1), and another four known analogs were isolated from L. alutacea. Compounds 1–4 were evaluated for cytotoxic activity against HL-60, MCF-7, A549, SW480, and SMMC-7721 cells and exhibited no appreciable activity against these tested cell lines with IC₅₀values above 40µM.

Experimental Section

General

Melting points (uncorrected): XT-4 melting point appa- ratus; [α]Dvalues: Jasco 20C digital polarimeter; UV spectra: UV-210A spectrometer;λmaxin nm; IR spectra: Bio-Rad FTS-135 spectrometer; 1D and 2D NMR spectra: DRX-400

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838 L.-J. Yanget al.·Iridoid Glycosides fromLagotis alutacea instrument; SiMe₄as internal reference,δin ppm,Jin Hz;

EI-MS: VG Autospec-3000 mass spectrometer.

Plant material

L. alutaceawas collected in Shangri-la Country, Yunnan Province, China, in November 2009. The plant was identified by Associate Prof. Qing-Song Yang, School of Chemical and Biological Technology, Yunnan Minzu University. A voucher specimen (09-1101) was deposited in the Key Laboratory of Ethnic Medicine Resource Chemistry, Yunnan Minzu Uni- versity, Yunnan, China.

Extraction and isolation

The dried whole plants (5 kg) were extracted four times with 95 % EtOH (4×20 L) at room temperature for 7 days, and the combined extracts were concentratedin vacuo. The residue was suspended in H2O, and then partitioned with petroleum ether (4×2 L), EtOAc (4×2 L) and n-BuOH (4×2 L), successively. The n-BuOH extract (436 g) was subjected to chromatography over silica gel, eluting with EtOAc-MeOH (80 : 1→0 : 1), to afford eight fractions (A–H). Fraction F (160 g) was purified by silica gel chromatography eluted with CHCl₃-MeOH (80 : 0 →0 : 1)

to give six fractions (1 – 6). Fr. 3 was repeatedly chro- matographed over a silica gel column eluting with CHCl3- MeOH (50 : 0→0 : 1), to yield compounds1(12 mg),2 (92 mg),3(16 mg), and4(150 mg).

Lagotisoside F(1). Colorless amorphous powder. M. p.:

155 – 157^◦C. [α]²⁴_D = −167.30 (c = 0.126, MeOH). – UV (MeOH): λmax(lgεmax) =222 (3.59), 303 (3.18) and 312 (3.74). – IR (KBr): ν = 3375, 1710, 1655, 1635, 1598, 1508, 1365, 1220, 1150, 1040, 830. – MS (EI, 70 eV): m/z = 698 [M]⁺, 683, 638 [M–2×OCH₃]⁺, 325, 207 [C6H3(OMe)2CH=CH-COOH–1]⁺, 147, 80. – HRMS ((+)-FAB): m/z=698.2425 (calcd. 698.2422 for C32H42O17, [M]⁺). –¹H and¹³C NMR (CD3OD): see Ta- ble1.

Acknowledgement

This work was supported by the Natural Science Foundation of China (21162042) and Yunnan Province (2010CD090), the Program for Innovative Research Teams (in Science and Technology) of the University of Yun- nan Province (IRTSTYN), and the Green Chemistry and Functional Materials Research for Yunnan Innovation Team (2011HC008).

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